DESCRIPTION (Taken from the application): The applicants broad, long-term research objective is to determine the biological function of neutrophils in skeletal muscle injury and regeneration. Neutrophils, the first inflammatory cell type to appear in injured muscle, have been suggested to exacerbate skeletal muscle injury following intense physical activity and muscle trauma. We have developed a cell culture model to test this hypothesis and have demonstrated that human neutrophils injure cultured human skeletal myotubes. The myotube injury was proportional to neutrophil concentration and to their state of activation. The proposed studies will extend our preliminary results by testing the following major hypotheses: 1) reactive oxygen and nitrogen intermediates (ROI and RNI, respectively) contribute to neutrophilmediated myotube injury, 2) injured myotubes stimulate ROI and RNI production from neutrophils, and 3) neutrophils exacerbate strain-induced myotube injury. The first hypothesis will be tested by manipulating the concentration of individual ROI and RNI using established procedures and by quantifying myotube injury. The second hypothesis will be evaluated by culturing neutrophils in the same medium that bathed myotubes during and following strain injury and assaying for ROI and RNI. The third hypothesis will be tested by mechanically stretching myotubes to induce minor myotube injury and then culturing them with neutrophils. Myotube injury will be quantified using a 51Cr release assay and will be qualitatively assessed via electron microscopy, using lanthanum as an extracellular tracer. Results from the proposed studies will provide novel information on the mechanism for neutrophil-mediated muscle injury and the ability of injured muscle to activate neutrophils to exacerbate muscle injury. With this information scientists can begin to systematically identify muscle-derived regulators of neutrophil function and develop therapeutic approaches to ameliorate muscle injury and enhance muscle regeneration following strenuous novel physical activity, muscle surgery, and a variety of inflammatory muscle diseases.